Supplemental material to this article can be found at:
http://molpharm.aspetjournals.org/content/suppl/2015/02/09/mol.114.095273.DC1
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MOLECULAR PHARMACOLOGY
Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/mol.114.095273
Mol Pharmacol 87:803–814, May 2015
Yet Another Function of p53—The Switch That Determines
Whether Radiation-Induced Autophagy Will Be Cytoprotective or
Nonprotective: Implications for Autophagy Inhibition as
a Therapeutic Strategy s
Shweta Chakradeo, Khushboo Sharma, Aisha Alhaddad, Duaa Bakhshwin, Ngoc Le,
Hisashi Harada, Wataru Nakajima, W. Andrew Yeudall, Suzy V. Torti, Frank M. Torti,
and David A. Gewirtz
Received August 6, 2014; accepted February 9, 2015
ABSTRACT
The influence of autophagy inhibition on radiation sensitivity was
studied in human breast, head and neck, and non–small cell lung
cancer cell lines, in cell lines that were either wild type or mutant/
null in p53, and in cells where p53 was inducible or silenced.
Whereas ionizing radiation promoted autophagy in all tumor cell
lines studied, pharmacological inhibition of autophagy and/or
genetic silencing of autophagy genes failed to influence sensitivity
to radiation in p53 mutant Hs578t breast tumor cells, HN6 head
and neck tumor cells, and H358 non–small cell lung cancer cells.
The requirement for functional p53 in the promotion of cytoprotective autophagy by radiation was confirmed by the observation
that radiation-induced autophagy was nonprotective in p53 null
H1299 cells but was converted to the cytoprotective form with
induction of p53. Conversely, whereas p53 wild-type HN30 head
Introduction
Virtually all patients with localized cancer are treated with
some combination of surgery, radiotherapy, and chemotherapy.
Therapy is often successful initially, but disease recurrence is
not uncommon and is often associated with resistance to treatment (Fodale et al., 2011). Although there are multiple mechanisms that could contribute to therapeutic resistance to radiation
such as enhanced DNA repair capacity and overexpression of
This research was supported in part by the National Institutes of Health
National Cancer Institute [Grants R01-CA171101 (to S.V.T. and F.M.T.) and
5R21-CA171974 (to D.A.G.)] and the National Institutes of Health National
Center for Advancing Translational Sciences [Award UL1-TR000058 (to D.A.G.)].
The Virginia Commonwealth University Flow Cytometry and Imaging Shared
Resource Facility is supported in part by the National Institutes of Health
National Cancer Institute [Grant P30-CA16059]. D.A.G. is also supported by the
American Cancer Society [Institutional Research Grant].
S.C. and K.S. contributed equally to this work.
dx.doi.org/10.1124/mol.114.095273.
s This article has supplemental material available at molpharm.aspetjournals.
org.
and neck cancer cells did show sensitization to radiation upon
autophagy inhibition, HN30 cells in which p53 was knocked down
using small hairpin RNA failed to be sensitized by pharmacological autophagy inhibition. Taken together, these findings indicate
that radiation-induced autophagy can be either cytoprotective
or nonprotective, a functional difference related to the presence
or absence of function p53. Alternatively, these findings could
be interpreted to suggest that whereas radiation can induce
autophagy independent of p53 status, inhibition of autophagy
promotes enhanced radiation sensitivity through a mechanism
that requires functional p53. These observations are likely to have
direct implications with respect to clinical efforts to modulate
the response of malignancies to radiation through autophagy
inhibition.
select survival signaling pathways, recent work has implicated
cytoprotective autophagy as a potential basis for resistance
that might be exploited for therapeutic purposes (Gewirtz,
2014a,b).
Studies in both cell culture and animal models have demonstrated the potential for improving the response to therapy
by the inhibition of cytoprotective autophagy through either
pharmacological intervention using drugs such as chloroquine or
genetic silencing of autophagy-related genes (Paglin et al., 2001;
Boya et al., 2005; Ito et al., 2005; Kondo et al., 2005; Abedin et al.,
2007; Amaravadi et al., 2007; Apel et al., 2008; Qadir et al., 2008;
Lomonaco et al., 2009; Carew et al., 2010; Wu et al., 2010; Ding
et al., 2011; Lopez et al., 2011; Shi et al., 2011; Tseng et al., 2011;
Wilson et al., 2011; Bristol et al., 2012; Godbole et al., 2012; Guo
et al., 2012; Liang et al., 2012; Rao et al., 2012). In addition, a
number of clinical trials have been initiated to determine whether
the chloroquine derivative, hydroxychloroquine, can be used to
ABBREVIATIONS: 3-MA, 3-methyladenine; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate;
PBS, phosphate-buffered saline; PI, propidium iodide; shRNA, small hairpin RNA.
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Departments of Medicine and Pharmacology and Toxicology, Massey Cancer Center (S.C., K.S., A.A., D.B., N.L., D.A.G.), and
Department of Oral and Craniofacial Molecular Biology, School of Dentistry (H.H., W.N., W.A.Y.), Virginia Commonwealth
University, Richmond, Virginia; and Departments of Molecular Biology and Biophysics (S.V.T.) and Medicine (F.M.T.), University
of Connecticut Health Sciences, Farmington, Connecticut
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Materials and Methods
T75 culture flasks were obtained from Cellstar (Monroe, NC).
Minimum essential medium-a containing L-glutamine was obtained
from Invitrogen (Grand Island, NY). Trypsin-EDTA (0.25% trypsin,
0.53 mM EDTA-4Na) and fetal bovine serum (FBS) were purchased
from Hyclone Scientific (Logan, UT) or Serum Source International
(Charlotte, NC). Terminal deoxynucleotidyl transferase–mediated
digoxigenin-deoxyuridine nick-end labeling assay reagents (terminal transferase, reaction buffer, and CoCl2 and fluorescein
dUTP) were purchased from Roche (Mannheim, Germany). Annexin
phosphatidylinositol and propidium iodide (PI) were purchased from
BD Biosciences (San Diego, CA). Trypan blue acetic acid, acridine
orange, formaldehyde, and 49,6-diamidino-2-phenylindole were purchased from Invitrogen (Eugene, OR). Phosphate-buffered saline (PBS),
L-glutamine, and penicillin-streptomycin were obtained from Gibco
(Life Technologies, NY). 5-Bromo-4-chloro-3-b-D-galactopyranoside) for
the b-galactosidase staining assay and M-PER mammalian protein
extraction reagent lysis buffer were purchased from Thermo Fisher
Scientific/Fermentas (Rockford, IL). Chloroquine, 3-methyladenine (3-MA),
NH4Cl, crystal violet and dimethylsulfoxide were obtained from SigmaAldrich(St. Louis, MO). The Annexin/apoptosis assay kit was obtained
from BD Biosciences. Bradford reagent was obtained from Bio-Rad
Laboratories (Hercules, CA).
Cell Lines. Hs578t mammalian breast tumor cells and A549 non–
small cell lung cancer cells were obtained from American Type
Culture Collection (Rockville, MD). The HN30, shHN30, and HN6
head and neck cancer cell lines were as previously described (Patel
et al., 2000). The H358 and A549 non–small cell lung cancer cell lines
were generously provided by Dr. Charles Chalfant from the Virginia
Commonwealth University Department of Biochemistry and Molecular
Biology. shHN30 and Hs578t shBECN1cells were maintained using
(1 mg/ml) puromycin. H1299 null and H1299 cells where p53 was
inducible by doxycycline were originally developed by Dr. Constantinos
Koumenis (Maecker et al., 2000). All cell lines were stored under liquid
nitrogen in 10% dimethylsulfoxide with 10% FBS.
Plasmid Construction and Virus Infection. Mission shRNA
bacterial stocks for ATG5 and BECN1 were purchased from SigmaAldrich (TRCN00151963 and TRCN0000299864, respectively). Lentiviruses were produced in HEK 293TN cells cotransfected using
Lipofectamine (Invitrogen, Carlsbad, CA) with psPAX2 and pMD2.G
packaging constructs (Addgene, Cambridge, MA). Viruses shed into
the media were then used to infect H1299 cells. Puromycin (1 mg/ml)
was used as the selection marker to enrich the infected cells.
Hs578t were also silenced for the Beclin-1 gene. Lentiviral shRNA
constructs from the laboratory of Dr. Hisashi Harada were transfected
into 293T cells with lentiviral packaging plasmid. The lentivirus shed
into the medium were collected to transfect Hs578t cells.
Cell Culture Conditions. Hs578t breast tumor cells were grown
in minimum essential medium-a supplemented with 20% FBS and
penicillin/streptomycin (0.5 ml/100 ml medium). HN30 and HN6 head
and neck cancer cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) containing 10% FBS, 2 mM L-glutamine, and 1%
penicillin-streptomycin. A549 non–small cell lung cancer cells were
cultured in 50% DMEM/50% of RPMI media and 1% penicillinstreptomycin. H358 non–small cell lung cancer cells were grown in
DMEM with 10% FBS and 1% penicillin-streptomycin. H1299 non–
small cell lung cancer cells were cultured in DMEM supplemented
with 10% FBS and 5% penicillin G/streptomycin; doxycycline at a
1mg/ml concentration was added for induction of p53 expression.
Cell cultures were maintained at 37°C under a humidified 5% CO2
atmosphere and examined for bacterial and fungal infections prior
to experiments.
Drug and Radiation Treatment. Chloroquine was prepared as
a stock solution of 50 mM in pure water and stored at 220°C. 3-MA
was prepared as a stock solution of 100 mM dissolved in PBS with
heating and refrigerated. NH4Cl was prepared as a 100-mM stock
in PBS. Chloroquine, 3-MA, and NH4Cl were added 3 hours before
radiation and cells were maintained in the presence of chloroquine,
3-MA, or NH4Cl until radiation was completed. Media were replenished
once treatment was terminated.
Hs578t cells were irradiated with 5 2 Gy over a period of 3 days.
HN30 and HN6 cells were treated with a single dose of 4 Gy, whereas
A549, H358, H1299 (in the absence of doxycycline [2Dox]), and H1299
(in the presence of doxycycline [1Dox]) cells were irradiated with a
single 6-Gy dose of radiation. The doses of radiation were chosen to
optimize the likelihood of detecting sensitization when autophagy was
inhibited.
Assessment of Cell Viability and Clonogenic Survival. Cell
viability was measured by trypan blue exclusion. Cells were incubated
with trypsin (0.25% trypsin-EDTA) for 5–10 minutes, stained with
trypan blue (0.4% trypan blue), and counted using a hemocytometer
with phase-contrast microscopy on the indicated days after radiation.
For the clonogenic survival assay, cells were plated in triplicate,
typically at a density of 100 cells/well. Ten to 14 days after treatment,
cells were washed with PBS, fixed with 100% methanol, air dried, and
stained with 0.1% (v/v) crystal violet. Cells in groups of 50 or more were
counted as colonies and data were normalized relative to untreated
controls.
Detection and Quantification of Autophagic Cells. Cells
were plated in six-well culture dishes, stained with acridine orange
at a final dilution of 1:10,000 in DMEM, and allowed to incubate at
37°C for 10 minutes. After two washes with PBS, cells were examined
and imaged under an inverted fluorescence microscope (Olympus,
Tokyo, Japan). All images were taken at the same magnification. For
quantification of autophagic vesicles, cells were trypsinized, harvested,
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improve the therapeutic response in a variety of malignancies
(Sotelo et al., 2006; Solomon and Lee, 2009).
Although studies in the literature generally support the
promotion of cytoprotective autophagy induced in response to
either chemotherapy or radiation, it is not clear that autophagy
uniformly has a protective function (Gewirtz, 2014a). In a
recent report, we demonstrated that chloroquine failed to
sensitize 4T1 murine breast tumor cells to radiation either in
cell culture or in a syngeneic animal model (Bristol et al., 2013),
which was also found to be the case for cisplatin (Maycotte
et al., 2012). Furthermore, genetic silencing of autophagy genes
failed to sensitize the 4T1 cells to radiation.
This work was designed to evaluate the impact of autophagy
inhibition on sensitivity to radiation in human tumor cell lines
derived from different tissues, specifically the triple negative
Hs578t human breast tumor cell line, HN6 and HN30 head and
neck cancer cells, and A549, H460, and H835 non–small cell
lung cancer cells. We find both cytoprotective and nonprotective
autophagy induced by radiation, with cytoprotective autophagy
occurring exclusively in cell lines with functional p53. Consistent
with this finding, radiation-induced autophagy was nonprotective
in p53 null H1299 non–small cell lung cancer cells but could
be converted to the protective form with induction of p53. Conversely, p53 wild-type HN30 head and neck cancer cells were
sensitized to radiation upon autophagy inhibition (cytoprotective
autophagy), whereas HN30 cells with small hairpin RNA
(shRNA)–mediated knockdown of p53 were refractory to such
sensitization (nonprotective autophagy).
This work suggests that clinical efforts to sensitize patients
to radiation (and possibly chemotherapy) through autophagy
inhibition may result in inconsistent and uninterpretable outcomes in the absence of information as to whether the autophagy
induced by treatment is cytoprotective or nonprotective, an
outcome that may be related to whether the tumor cells are
wild type or mutant in p53.
p53, Autophagy, and Radiation in Cancer Therapy
and washed with PBS. Pellets were resuspended in PBS, stained with
a 1:10,000 dilution of acridine orange for 10 minutes, and analyzed by
BD FACSCanto II using BD FACSDiva software at the Virginia
Commonwealth University Flow Cytometry Core Facility. A minimum
of 10,000 cells within the gated region were analyzed.
Flow Cytometry Analysis Using Annexin V–PI Staining to
Determine Apoptotic and Necrotic Cells. Treated cells were
collected and labeled fluorescently using a fluorescein isothiocyanate
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(FITC) Annexin V apoptosis detection kit I. Labeling was performed
by adding 500 ml of binding buffer, 5 ml Annexin V–FITC, and 5ml
propidium iodide to each sample (FITC Annexin V apoptosis detection
kit; BD Biosciences, San Jose, CA). Samples were mixed gently and
incubated in the dark for 15 minutes. Annexin V–PI labeled cells were
measured by flow cytometry and analyzed by BD FACS Canto II and
BD DIVA software. A minimum of 10,000 cells were counted within
the gated region.
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Fig. 1. Induction of autophagy by radiation in Hs578t breast tumor cells; autophagy inhibition by chloroquine does not result in sensitization. (A)
Hs578t cells were exposed to 5 2 Gy of radiation over a period of 3 days and the viable cell number was monitored by trypan blue exclusion. Data
represent the mean of three experiments 6 S.E. (B) Induction of autophagy upon irradiation of Hs578t cells. Acridine orange staining of autophagic
vacuoles on day 7 after exposure to 5 2 Gy of radiation with and without chloroquine (25 mM). (C) Quantification of autophagy measured by flow
cytometry. Data are representative of the mean 6 S.E. of three experiments. (D) p62/SQSTM1 degradation indicative of autophagic flux in Hs578t cells
on day 1 after radiation. Serum starvation was included as a positive control. The experiment was reproduced three times. (E) Clonogenic survival assay
in Hs578t cells. Data represent the average of three experiments plotted with mean 6 S.E. (F) Induction of apoptosis in irradiated cells with or without
chloroquine. *P , 0.05 compared with control. Con, control; CQ, chloroquine; IR, radiation.
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Western Blot Analysis. After the indicated treatments, cells were
scraped from the culture dishes, collected, and lysed using M-PER
mammalian protein extraction reagent (Thermo Fisher Scientific,
Waltham, MA) containing protease and phosphatase inhibitors. Protein
concentrations were determined by the Bradford assay (Bradford, 1976)
(Bio-Rad Laboratories). Total protein was then diluted in SDS sample
buffer and dry boiled for 10 minutes. Protein samples were subjected to
SDS-PAGE, transferred to polyvinylidene difluoride membrane, and
blocked in 5% milk/1 PBS/ 0.1% Tween for 1 hour. Primary antibodies
used at a 1:1000 dilution with overnight incubation were p62 (BD
Transduction), ATG5 (Cell Signaling Technology, Danvers, MA), BECN1
(Cell Signaling Technology), LC3II (Cell Signaling Technology), p53 (BD
Pharmingen), and b-actin (Santa Cruz Biotechnology). The membrane was
then incubated with secondary antibody of either horseradish peroxidase–
conjugated goat anti-rabbit IgG antibody (1:5000; Sigma-Aldrich) or goat
anti-mouse (1:5000; Sigma-Aldrich) for 1 hour, followed by extensive
washing with Tween-PBS. Blots were developed using Pierce enhanced
chemiluminescence reagents and Bio-Max film (Thermo Fisher Scientific).
Statistical Analysis. Statistical differences were determined by
StatView statistical software (SAS Institute, Cary, NC). The data
were expressed as means 6 S.E. Comparisons were made using oneway analysis of variance followed by the Bonferroni post hoc test.
P values ,0.05 were taken as statistically significant.
Results
Radiation Induces Autophagy and Prolonged Growth
Arrest in Hs578t Breast Tumor Cells. Previous studies from
this laboratory have shown that radiation induces cytoprotective
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Fig. 2. Inhibition of radiation-induced autophagy in Hs578t cells using
NH4Cl does not result in sensitization. (A) Influence of ammonium chloride
(10 mM) on autophagic vacuole formation based on acridine orange staining
(day 4 after radiation). (B) p62/SQSMT1 degradation indicative of autophagic
flux in Hs578t cells (day 1 after radiation). The experiment was repeated three
times with similar results. (C) Clonogenic survival assay indicating that
inhibition of autophagy by NH4Cl failed to sensitize Hs578t cells to radiation.
Data are representative of three experiments plotted with mean 6 S.E. *P ,
0.05 compared with control. Con, control; IR, radiation.
Fig. 3. Inhibition of radiation-induced autophagy in Hs578t cells using
3-MA does not result in sensitization. (A) Influence of 3-MA (5 mM) on
autophagic vacuole formation based on acridine orange staining (day 1
after radiation). (B) p62/SQSMT1 degradation indicative of autophagic
flux in Hs578t cells (day 1 after radiation). The experiment was repeated
three times with similar results. (C) Clonogenic survival assay indicating
that inhibition of autophagy by 3-MA failed to sensitize Hs578t cells to
radiation. Data are the average of three experiments plotted with mean 6
S.E. *P , 0.05 compared with control. IR, radiation.
p53, Autophagy, and Radiation in Cancer Therapy
Annexin V/PI indicated that only between 15 and 20% of
the cell population was undergoing apoptosis after radiation.
Furthermore, inhibition of autophagy using chloroquine did
not increase the extent of apoptosis, which supports the
conclusion that autophagy is unlikely to be cytoprotective in
this experimental system.
Chloroquine Fails to Sensitize Hs578t Cells to a Single
Dose of Radiation. Because the 5 2 Gy radiation alone
produced a relatively pronounced reduction in clonogenic survival,
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autophagy in MCF-7 and ZR-75 breast tumor cell lines (Wilson
et al., 2011; Bristol et al., 2012), both of which are wild type in
p53. The first series of experiments presented here involves
Hs578t breast tumor cells, which are mutant in p53 (NievesNeira and Pommier, 1999). Figure 1A indicates that exposure of
Hs578t breast tumor cells, which are triple negative cells
(Foulks et al., 2010), to radiation (5 2 Gy over a period of
3 days) results primarily in growth arrest; the growth arrest was
relatively prolonged as the cells did not recover for at least
11 days after irradiation (data not shown).
Figure 1B indicates that radiation promoted autophagy in
the Hs578t cells based on the detection of puncta by acridine
orange staining. Figure 1C (in which the extent of autophagic
vesicle formation was assessed by flow cytometry) indicates that
once autophagy was induced, its extent was relatively stable
over a period of 7 days (with a slight transient increase at
day 5).
Evidence That Radiation-Induced Autophagy Does
Not Have a Cytoprotective Function in Hs578t Breast
Tumors Based on Lack of Sensitization by the Pharmacological Autophagy Inhibitor, Chloroquine. To determine whether radiation-induced autophagy has a cytoprotective
function in Hs578t cells, autophagy was inhibited with the use of
three separate pharmacological inhibitors: 3-MA, chloroquine,
and NH4Cl. Appropriate concentrations (25 mM for chloroquine,
5 mM for 3-MA, and 10 mM for NH4Cl) were chosen by determining
concentrations that were minimally toxic to the tumor cells using
an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (data not shown). Figure 1B confirms that chloroquine inhibited
radiation-induced autophagy based on the change from red/
orange to yellow of the autophagosomes, which reflects the fact
that chloroquine interferes with the acidification required for
fusion of lysosomes with the autophagosome (Bristol et al.,
2012). Additional evidence that radiation promotes autophagy
in the Hs578t cells, as well as that chloroquine inhibited radiationinduced autophagy, is provided by the observation that
chloroquine prevented the radiation-induced degradation of
p62/SQSTM1 (Fig. 1D), an indication of radiation-induced
autophagic flux (Bjørkøy et al., 2009; He and Klionsky, 2009;
Feng et al., 2014). Serum starvation was used as a positive
control.
To determine whether autophagy might be playing a cytoprotective (or alternatively a cytotoxic) function in the Hs578t
cells, clonogenic survival was assessed in cells exposed to radiation
in the absence and presence of chloroquine. As shown in Fig. 1E,
only an apparent additive effect of radiation and chloroquine was
observed; that is, the reduced clonogenic survival in cells treated
with the combination of radiation and chloroquine reflects the
combined antiproliferative and/or cytotoxic effects of each of the
treatment modalities. Specifically, with exposure of Hs578t cells
to fractionated radiation alone, there is an approximately 55%
reduction in survival, whereas with chloroquine, cell survival is
reduced by approximately 30%. The combination treatment of
radiation plus chloroquine reduced survival by approximately
85%, which is what would be anticipated from additive toxicities of
the individual treatments.
Blocking Autophagy with Chloroquine Does Not
Promote Apoptosis in Irradiated Hs578t Cells. In classic
cytoprotective autophagy, cells use autophagy as a mechanism
for cell survival and consequently when autophagy is inhibited,
cells die, primarily by apoptosis (Gewirtz et al., 2009). As shown
in Fig. 1F, assessment of apoptosis by flow cytometry using
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Fig. 4. Genetic inhibition of radiation-induced autophagy in Hs578t cells
does not result in sensitization. (A) Autophagic vacuole formation based on
acridine orange staining in irradiated Hs578t cells in which Beclin-1 was
silenced (day 1 after radiation). (B) p62/SQSMT1 degradation indicative of
autophagic flux in Hs578t cells (day 1 after radiation). The experiment
was repeated three times with similar results. (C) Clonogenic survival
assay indicating that silencing of Beclin-1 failed to sensitize Hs578t cells
to radiation. The inset shows Beclin-1 silencing. Data represent the average
of three experiments plotted with mean 6 S.E. *P , 0.05 compared with
control. IR, radiation.
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it might be argued that the studies lack sufficient sensitivity to
detect sensitization. Consequently, the clonogenic survival
studies were repeated using a single radiation dose of 4 Gy.
Chloroquine also failed to sensitize the Hs578t cells to the 4 Gy
of radiation in that the reduction in clonogenic survival for the
combination treatment (approximately 52%) essentially reflected the additive toxic effects of the chloroquine (approximately 13%) and the radiation (approximately 38%) (data not
shown).
Evidence That Radiation-Induced Autophagy Does
Not Have a Cytoprotective Function in Hs578t Breast
Tumor Cells Based on Lack of Sensitization by the Pharmacological Autophagy Inhibitors, Ammonium Chloride
and 3-MA. Figure 2 presents studies that confirm that radiation
promotes a nonprotective form of autophagy in Hs578t breast
tumor cells by assessing the influence of another late stage
autophagy inhibitor, NH4Cl (Ikeda et al., 2013), on sensitivity to
radiation. Figure 2A shows the promotion of autophagy by
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Fig. 5. Radiation-induced autophagy in head and neck cancer cells. Inhibition of radiation-induced autophagy sensitizes p53 wild-type HN30 but not
p53 mutant HN6 head and neck cancer cells. (A and B) Acridine orange staining of autophagic vacuoles (day 1 after exposure to 4 Gy of radiation) in
HN30 cells (A) and HN6 cells (B) with and without chloroquine (10 mM). (C and D) p62/SQSMT1 degradation indicative of autophagic flux in HN30 cells
(C) and HN6 cells (D) day 1 after radiation. (E) Clonogenic survival assay indicating sensitization upon inhibition of radiation-induced autophagy in
HN30 cells. Data represent the average of three experiments plotted with mean 6 S.E. (F) Clonogenic survival assay indicating lack of sensitization
upon inhibition of radiation-induced autophagy in HN6 cells. *P , 0.05 compared with control; **P , 0.05 compared with radiation. CQ, chloroquine; IR,
radiation.
p53, Autophagy, and Radiation in Cancer Therapy
radiation and that NH4Cl inhibits radiation-induced autophagy
based on the change from the reddish-orange color of the acridine
orange–stained vesicles to a yellowish color (again indicative of
interference with the acidification step required for completion of
autophagy). Figure 2B again indicates that radiation reduced
p62/SQSTM1 levels, and that NH4Cl interfered with the degradation of p62. In the clonogenic survival assay presented in
Fig. 2C, the combination of an autophagy inhibitor, NH4Cl, with
radiation showed a modest but insignificantly greater effect than
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radiation alone that again, appears to reflect the additive
antiproliferative effects of radiation and NH4Cl.
Additional studies were performed using the early stage
autophagy inhibitor, 3-MA (Boya et al., 2005). Figure 3A
demonstrates that, as would be expected, there is a decline in
autophagic vesicle formation in Hs578t cells treated with 3-MA
and radiation (in contrast with the accumulation of vacuoles in
the studies with the late stage autophagy inhibitors chloroquine
and ammonium chloride). The autophagic flux in irradiated
cells is shown by the degradation of p62/SQSMT1 in Fig. 3B,
which is prevented by 3-MA. The clonogenic survival assay
presented in Fig. 3C indicates that 3-MA failed to alter sensitivity
to radiation, confirming the findings when autophagy was
inhibited using either chloroquine or NH4Cl.
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Fig. 6. Evidence for p53 dependence of cytoprotective autophagy; lack of
sensitization by chloroquine in HN30 cells with knockdown of p53. (A)
Acridine orange staining in shp53 HN30 shows induction of autophagy
and inhibition of autophagy by chloroquine. (B) p62/SQSMT1 degradation
indicative of autophagic flux in shp53HN30 cells (day 1 after radiation).
(C) Clonogenic survival assay in shp53 HN30 cells indicating lack of
sensitization by autophagy inhibition. CQ, chloroquine; IR, radiation.
*P , 0.05 compared with control.
Fig. 7. Inhibition of radiation-induced autophagy sensitizes A549 non–
small cell lung cancer cells. (A) Induction of autophagy upon radiation in
A549 cells. Acridine orange staining of autophagic vacuoles (day 1 after
exposure to 6 Gy of radiation) with and without chloroquine (10 mM). (B)
p62/SMQST1 degradation indicative of autophagic flux in A549 cells. The
experiment was repeated three times with similar results. (C) Clonogenic
survival studies indicative of sensitization. Data represent the average of
three experiments plotted with mean 6 S.E. *P , 0.05 compared with
control; **P , 0.05 compared with radiation alone. CQ, chloroquine; IR,
radiation.
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Autophagy Inhibition with Chloroquine Fails to
Sensitize HN30 Cells with Knockdown of p53. To this
point, all of the data presented are essentially circumstantial
in terms of implicating a requirement for functional p53 in the
capacity of ionizing radiation to promote the cytoprotective
form of autophagy. To more directly address this question,
p53 was knocked down in the p53 wild-type HN30 head and
neck tumor cells (shown in the inset in Fig. 6C). Supplemental
Figure 2B again shows sensitization in the clonogenic survival
assay in which autophagy in the HN30 cells was inhibited
by chloroquine (as supported by the acridine orange staining
in Fig. 5C and Supplemental Fig. 2B). This study had to be
repeated for comparative purposes with the p53 silenced cells
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Evidence That Radiation-Induced Autophagy Does
Not Have a Cytoprotective Function in Hs578t Breast
Tumors Based on Lack of Sensitization by Genetic
Autophagy Inhibition. Although the results generated
using three separate pharmacological autophagy inhibitors
were consistent in failing to demonstrate significant sensitization to radiation, it was nevertheless necessary to confirm
these observations through genetic inhibition of autophagy.
The inset in Fig. 4C indicates that Beclin-1, often considered
as an essential gene in the autophagy pathway (Kang et al.,
2011), was knocked down using an shRNA strategy. Figure 4A
shows that induction of autophagy by radiation was suppressed in the Beclin-1–silenced cells based on acridine orange
vacuole staining. Figure 4B further demonstrates that the
silencing of Beclin-1 interfered with the radiation-induced
degradation of p62/SQSMT1. Somewhat disconcertingly, silencing of Beclin-1 had only a modest effect on degradation of
p62/SQSMT1 under conditions of serum starvation, suggesting
that autophagy under these conditions may not be entirely
Beclin-1 dependent. Figure 4C shows that when Hs578t cells
were silenced for Beclin-1, radiation sensitivity was identical to
that in the Hs578t cells without Beclin-1 silencing. These studies
appear to confirm the conclusion that autophagy induced by
radiation in Hs578t cells is not protective in function.
Radiation-Induced Autophagy Is Cytoprotective in
p53 Wild-Type HN30 Head and Neck Cancer Cells and
Nonprotective in p53 Mutant HN6 Head and Neck
Cancer Cells. We previously reported on the cytoprotective
autophagy induced by radiation in MCF-7 and ZR-75 breast
tumor cell lines (Wilson et al., 2011; Bristol et al., 2012) and
the data presented above establish that autophagy induced by
radiation can likewise be nonprotective in breast tumor cells.
To extend these findings to tumor cells derived from another
tissue source, studies were conducted in p53 wild-type HN30
and p53 mutant HN6 head and neck tumor cells, a disease in
which radiation is also a conventional therapy (Zackrisson
et al., 2003). Figure 5A again shows radiation-induced autophagy
in the HN30 cells based on acridine orange staining and the
capacity of chloroquine to alter the nature of the staining, indicative of a blockade to the acidification step that is necessary
for autophagosome-lysosome fusion. Figure 5C confirms that
radiation is promoting degradation of p62/SQSTM1 and that this
degradation is essentially abrogated by chloroquine. Figure 5E
presents clonogenic survival in the HN30 cells with radiation
alone and radiation plus chloroquine. Although the clonogenic
survival studies presented in Fig. 5E are suggestive of sensitization, we recognize that it could be argued that the data in are
merely showing additive toxicities of the combination treatment.
To demonstrate that chloroquine actually sensitized the HN30
cells to radiation, temporal response studies were performed. As
shown in Supplemental Fig. 1, the combination of chloroquine
with radiation produces a sustained growth arrest that is not
seen for either radiation or chloroquine treatment alone.
Similar studies performed in the HN6 cells show the induction
of autophagy by radiation and interference with autophagy by
chloroquine (Fig. 5B). Furthermore, chloroquine is again shown
to prevent the degradation of p62 induced by radiation (Fig. 5D).
However, in contrast with the findings with the HN30 cells,
Fig. 5F shows a lack of radiation sensitization by chloroquine in
the HN6 cells by the clonogenic survival assay. This lack of
sensitization was confirmed by the temporal response study
presented in Supplemental Fig. 1B.
Fig. 8. Inhibition of radiation-induced autophagy fails to sensitize H358
non–small cell lung cancer cells. (A) Induction of autophagy upon irradiation.
Acridine orange staining of autophagic vacuoles (day 1 after exposure to
6 Gy of radiation) with and without chloroquine (10 mM). (B) p62/SQSMT1
degradation indicative of autophagic flux in H358 cells. (C) Clonogenic
survival studies indicative of lack of sensitization. Data represent the
average of three experiments with mean 6 S.E. CQ, chloroquine; IR,
radiation. *P , 0.05 compared with control.
p53, Autophagy, and Radiation in Cancer Therapy
Radiation-Induced Autophagy Is Nonprotective in
p53 Null H1299 Non–Small Cell Lung Cancer Cells but
Is Converted to Protective Autophagy with Induction
of p53. Taken together, these findings are clearly suggestive
of p53 dependence for radiation-induced cytoprotective autophagy
since in this and previous work, protective autophagy was observed in cells with functional p53 (MCF-7, ZR-75, A549, H460,
and HN30) but not in cells null or mutant in p53 (4T1, Hs578t,
H358, and HN6). This conclusion is supported by the findings
in the HN30 cells with knockdown of p53, shown above. To
more convincingly establish this conclusion, additional studies
were designed to assess the impact of interfering with radiationinduced autophagy in p53 null H1299 non–small cell lung
cancer cells and their isogenic counterparts in which p53 was
induced by doxycycline. Figure 9C indicates that radiation
induced autophagy in both the p53 null cells and the cells
where p53 was induced by doxycycline (the Western blot in
Fig. 9A indicates p53 induction). Figure 9, C and D, present
evidence that autophagy has been inhibited based on a decrease in acidic vesicle formation when ATG5 is knocked down
(knockdown indicated in Fig. 9B) or an accumulation of LC3II
with exposure to chloroquine (Fig. 9D). Radiation sensitivity
was essentially unaltered when radiation-induced autophagy
in H1299 cells that are null in p53 was inhibited (either by
silencing of ATG5 or chloroquine treatment) (Fig. 10, A and C),
with the exception of a small transient increase on day 1
(shown in Fig. 10A). In quite dramatic contrast, both ATG5
silencing and treatment with chloroquine resulted in pronounced
Fig. 9. Evidence for p53 dependence of cytoprotective autophagy;
induction of p53 results in sensitization to radiation when
autophagy is inhibited in H1299 non–small cell lung cancer
cells. (A) Induction of p53 under the influence of doxycycline
in H1299 cells. (B) Knockdown of ATG5 in both H1299 null
(2Dox) and p53 inducible (1Dox) cell lines. (C) Induction of
autophagy (upper panel) as assessed by acridine orange
staining in H1299 cells and inhibition of autophagy (lower
panel) when the ATG5 gene is knocked down as shown by
reduction in acidic vesicle formation. (D) Accumulation of
LC3II when the autophagy inhibitor chloroquine is used in
combination with radiation indicative of inhibition of autophagic
flux in both H1299 (2Dox) and (1Dox) cells. CQ, chloroquine;
Dox, doxycycline; IR, radiation; shCont, scrambled control.
Original magnification, 20.
Downloaded from molpharm.aspetjournals.org at ASPET Journals on July 28, 2017
since the experiments presented in Fig. 5 had been performed
months previously. By contrast, the shp53 HN30 cells failed to
be sensitized to radiation upon autophagy inhibition (Fig. 6C).
Again, the inhibition of autophagy was confirmed based on
acridine orange staining and interference with the degradation of p62/SMQST1 (Fig. 6, A and B).
Radiation-Induced Autophagy Is Protective in p53
Wild-Type A549 Non–Small Cell Lung Cancer Cells and
Nonprotective in p53 Mutant H358 Non–Small Cell
Lung Cancer Cells. In a recent report, radiation was shown
to promote cytoprotective autophagy in A549 and H460 non–
small cell lung cancer cells (Ko et al., 2014), both of which are
wild type in p53. We have confirmed these findings in A549
cells (H460 cells not shown). Figure 7A shows the induction of
autophagy by ionizing radiation and interference with autophagy
by chloroquine based on the yellow color that reflects suppression
of acidification and autophagosome-lysosome fusion. Figure 7B
shows that the p62/SQSTM1 degradation is compromised by
the chloroquine treatment. Figure 7C demonstrates sensitization to radiation by the chloroquine based on a count of viable
cells.
When similar studies were performed using the (p53 mutant)
H358 non–small cell lung cancer cell line, autophagy and
autophagic flux were again induced by radiation and were
inhibited by chloroquine (Fig. 8, A and B). However, in contrast with the results observed in the A549 and H460 cells,
chloroquine failed to alter radiation sensitivity in the H358
cells (Fig. 8C).
811
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Chakradeo et al.
sensitization to radiation in the H1299 cells when p53 was
induced by doxycycline (Fig. 10, B and D). Control growth and
growth in the presence of chloroquine alone were purposely
omitted from Fig. 10, A–D, to limit the range of the y-axis and
allow focus on the effects (or lack thereof) of autophagy inhibition.
Chloroquine alone had minimal impact on cell growth. Baseline
apoptosis and apoptosis in the presence of chloroquine are also not
shown in Fig. 10, E and F, since apoptosis was essentially
undetectable in the absence of irradiation of the cells. Furthermore, induction of apoptosis was observed in H1299 (with
induction by doxycycline [1Dox]) cells but not the H1299 (in the
absence of doxycycline [2Dox]) cells upon inhibition of autophagy
by either chloroquine or genetic knockdown of ATG5 as shown in
Fig. 10, E and F.
Discussion
Fig. 10. Evidence for p53 dependence of cytoprotective
autophagy; induction of p53 results in sensitization to
radiation when autophagy is inhibited in H1299 non–
small cell lung cancer cells. (A and C) H1299 non–small
cell lung carcinoma cells null in p53 do not show any
change in sensitivity to radiation when autophagy is
blocked using chloroquine or genetic knockdown of ATG5. (B
and D) Induction of p53 under the influence of doxycycline
results in sensitization to radiation when autophagy is
blocked using chloroquine or knockdown of ATG5. (E and F)
Induction of apoptosis in H1299 (+Dox) cells when autophagy
is blocked using chloroquine or genetic knockdown of ATG5
(n = 3, mean 6 S.E.). *P , 0.05; #P , 0.001. CQ, chloroquine;
Dox, doxycycline; IR, radiation; shCont, scrambled control.
Downloaded from molpharm.aspetjournals.org at ASPET Journals on July 28, 2017
It is generally thought that when chemotherapy and
radiation promote autophagy in tumor cells, the autophagy
has a cytoprotective function that can be inhibited to sensitize
the cells to treatment. We and our collaborators recently
demonstrated lack of sensitization to radiation as well as to
cisplatin in the 4T1 murine breast cancer model upon autophagy
inhibition, indicating the existence of a nonprotective function
of autophagy in tumor cells (Maycotte et al., 2012; Bristol et al.,
2013).
In this work, we demonstrate a lack of sensitization to
radiation in Hs578t breast cancer cells upon inhibition of
radiation-induced autophagy utilizing both multiple pharmacological approaches as well as confirming these results with
genetic silencing of Beclin-1. The concept that inhibition of
autophagy fails to sensitize Hs578t cells to radiation therapy
is supported by the lack of increased apoptosis when autophagy
is inhibited, which supports the absence of cytoprotective
autophagy. At the same time, the autophagy does not appear to
demonstrate a cytotoxic or growth inhibitory function in this
model system since there is no reduction in radiation sensitivity
when autophagy is inhibited. Parallel studies demonstrate the
existence of both cytoprotective and nonprotective autophagy,
respectively, in the A549/H460 and H358 non–small cell lung
cancer cells and in the HN30 and HN6 head and neck cancer
cells. Thus, the concept that radiation can produce either
cytoprotective or nonprotective autophagy is supported by
studies in human tumor cells derived from different tissues.
p53, Autophagy, and Radiation in Cancer Therapy
radiation-induced cytoprotective and nonprotective autophagy
in isogenic tumor cell lines.
The potential importance of this work relates, in large part,
to the fact clinical trials are in progress to study whether
sensitivity to chemotherapy or radiation treatment can be
increased by using the autophagy inhibitors chloroquine or
hydroxychloroquine. Our findings indicate that the fact that
radiation can induce autophagy in a given tumor cell does not
obligatorily mean that this autophagy is protective. Consequently, if the strategy of autophagy inhibition is to potentially
be successful as a therapeutic strategy, it will likely be necessary
to stratify patients in terms of whether the autophagy being
induced is cytoprotective in function.
Acknowledgments
The authors thank the Virginia Commonwealth University Flow
Cytometry and Imaging Shared Resource Facility for conducting
quantification of PI staining for cell cycle analysis, Annexin V–PI
staining, and acridine orange staining.
Authorship Contributions
Participated in research design: Chakradeo, Sharma, Alhaddad,
Bakhshwin, Le, Harada, Nakajima, Yeudall, Gewirtz.
Conducted experiments: Chakradeo, Sharma, Alhaddad, Bakhshwin,
Le, Nakajima.
Contributed new reagents or analytic tools: S. V. Torti, F. M. Torti.
Performed data analysis: Chakradeo, Sharma, Alhaddad, Bakhshwin,
Le, Harada, Nakajima, Yeudall, Gewirtz.
Wrote or contributed to the writing of the manuscript: Chakradeo,
Sharma, Alhaddad, Bakhshwin, Le, Harada, Yeudall, Gewirtz.
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